We have obtained physiological and genetic evidence that RNA polymerase (or certain of its subunits) is involved in (p)ppGpp biosynthesis in Bacillus subtilis. This will be tested by in vitro studies with purified RNA polymerase. A new class of relaxed mutants have been identified and mapped in this bacterium. We propose to characterize them biochemically by examining (p)ppGpp synthesis in vitro. We have previously shown that limitation for certain aminoacyl-tRNA species elicits allele specific phenotypic suppression of some frameshift and nonsense alleles of lacZ and rIIB. We propose to extend these studies, particularly in the rIIB system, where the nucleotide sequence is known. Our aim is to correlate the specificities of phenotypic suppression -- that is, which aminoacyl-tRNA limitations suppress which mutants -- with the codons specified in the vicinity of the suppressible allele. This will be done both by comparing the behavior of different suppressible alleles, and by deliberately modifying them. In the case of phenotypic suppression of nonsense alleles, we will quantitate the production of full length lacZ protomers by gel electrophoresis and a radioimmunological assay. We have found that when an MS2 in vitro translation system is starved for cysteine, tyrosine, or tryptophan, premature chain termination at specific sites in coat and synthetase proteins is induced. We will characterize this phenomenon in more detail.